Restriction endonucleases are enzymes which recognize specific short sequence of DNA and cleave the DNA in both the strands (Alfred,2004). Werner Arber in 1960s predicted that the restriction enzyme act on a DNA sequence, called a recognition site. In 1970s, Hamilton Smith purified a restriction enzyme from Haemophilia influenzae(H.infuenzae) and showed that this enzyme cut DNA at specific sequence. The sequence is usually six base pair long. The cutting of DNA molecule into smaller pieces is known as restriction digestion. Smith showed that, when this recognition site is present in H.infuenzae it does not cut the host cell DNA (Heidi Chial, n.d) . There are four different types of restriction endonucleases. They are Type I restriction endonuclease, Type II restriction endonuclease, Type III restriction endonuclease and Type IV restriction endonuclease (Heidi Chial, n.d).
Restriction endonucleases were primarily used for obtaining DNA fragments for agarose and polyacrylamide gel electrophoresis. Later, restriction enzymes became the workhorse of molecular biology. DNA mapping uses restriction endonuclease. Mutants were made by deleting one or more fragments of DNA. Early days of sangers DNA sequencing made use of restriction enzymes to cut DNA. The doorway to recombinant DNA technology began with a work done using restriction endonuclease and SV40 DNA. SV40 was converted to a vector for recombinant DNA( Roberts,2005).
Type II restriction endonucleases are the most common restriction enzymes. Type II cut DNA within their recognition sites. The recognition sites are palindromic (R.W.Old,1980). EcoRI is a type II restriction endonuclease. They recognize palindromic sequences that are 4 to 8 base pairs(bp) long and cut DNA within the recognition site in the presence of Mg2+ (Alfred,2001). EcoRI recognizes the sequence GAATTC. EcoRI name originates from the species the enzyme was isolated, Escherichia coli (E.coli), R denotes the strain RY13 and I represents it is the first enzyme to be isolated from Escherichia coli RY13. (R.W.Old,1980).
EcoRI was used to cut lambda(?)DNA and pUC19 DNA. Lambda was discovered by Esther Lederberg in 1951 (Casjens.S,2015). Bacteriophage lambda infects E.coli. This infection increases the rate of genetic recombination as a part of infection cycle (Hillyar.C,2012). When the DNA is isolated from the phage particle, it is in the form of a linear duplex molecule. The DNA is 48.5 kb. Lambda DNA forms a circular structure when inserted into the host cell. This is because of the 5′-projections of 12 nucleotides. These projections form the cos site (R.W.Old,1980). Lambda DNA was used as the ladder to determine the molecular weight of fragmented pUC19 DNA.
pUC19 is a cloning vector. It is small, circular and double-stranded. pUC19 plasmid has multiple cloning site within the coding region of LacZ fragment. Restriction site for EcoRI is present within the 54bp multiple cloning site. pUC19 has only one recognition site for EcoRI (Souza Xavier,2009). It has high copy number. pUC19 is commonly used cloning vector that convey tetracycline(Tet) and Ampicillin(Amp) resistance(New Bio Labs,2018).
Fig:1.1 Restriction sites of pUC19 vector( New Bio Labs,2018)
Gel electrophoresis is a technique ud to separate DNA molecules based on size. Agarose is a plant polysaccharide that is used to make a semi-solid gel for gel electrophoresis of DNA(Malacinski,2005). When electric field is applied, DNA migrates to the positive electrode. Larger DNA fragments migrate with a slower speed. Over several hours, DNA fragments migrate over the gel , forming a ladder of distinct bands. Each band contain several DNA molecules of same length(Alberts,2015).
The aim of the experiment is to examine and distinguish plasmid DNA by restriction endonuclease digestion .